In the yeast Saccharomyces cerevisiae, the RAD3 gene is required for the excision of pyrimidine dimers formed in UV-damaged DNA (Freidberg, E. et al., in “DNA repair and Mutagenesis” pp. 233–316, ASM Press Washington, D.C., 1995; Siede, W. in “DNA Damage and Repair Vol. 2 ” pp. 307–333, Ed. Nickoloff J. A. and Hoekstra M. F., Humana Press, Totowa, N.J., 1998). In addition to the involvement of RAD3 in excision repair, the yeast RAD3 gene is also essential for cell viability (Freidberg, E. et al., in “DNA repair and Mutagenesis” pp. 233–316, ASM Press Washington, D.C., 1995; Siede, W. in “DNA Damage and Repair Vol. 1, Part II” pp. 307–333, Ed. Nickoloff J. A. and Hoekstra M. F., Humana Press, Totowa, N.J., 1998). The RAD3 gene encodes a protein, Rad3, consisting of 778 amino acids (˜90 kDa) and having 20 predominantly acidic amino acids at the carboxyl terminus. Deletions of this acidic region have no obvious effect on cell viability or DNA repair (Reynolds, et al., Nucleic Acid Res. 13: 2357–2372, 1985).
The yeast Rad3 protein is a single stranded DNA dependent ATPase (Sung, P. et al., Proc. Nat. Aced. Sci. 84:6045–6049, 1987). It is also an ATP-dependent DNA helicase with 5′–3′ specificity (Sung, P. et al., Proc. Nat. Aced. Sci. 84:8951–8955, 1987). Purified yeast Rad3 catalyzes the displacement of RNA fragments annealed to complementary DNA and possesses a potent helicase activity against DNA:RNA hybrid duplexes. The ATP-hydrolysis reaction is not affected by ribonucleotide homopolymers (Bailly V. et al., Proc. Nat. Aced. Sci. 88:9712–9716, 1991; Naegeli, H. et al., J. Biol. Chem. 267:7839–7844, 1992). Purified Rad3 exhibits preferential binding to UV-damaged DNA over non-damaged DNA. This binding is dependent on ATP hydrolysis and is promoted by negative superhelicity (Sung, P. et al., J. Biol. Chem. 269:8303–8308, 1994).
Recently, Guzder et al. showed an involvement of the yeast RAD3 gene in transcription by RNA polymerase II (Guzder, S. et al., Nature 367:91–94, 1994). Biochemical and genetic analysis has shown that Rad3 is an authentic subunit of transcription factor b or Tfb, also known as transcription Factor IIH or TFIIH (Feaver W J et al., Cell 7:1379–1387, 1993; Bardwell, L. et al., Proc. Natl. Aced. Sci. 91:3926–3930, 1994). Further in vitro reconstitution studies using recombinant proteins have established that the helicase activity of Rad3 and other TFIIH subunits are required for the incision step of nucleotide excision repair (Sung, P. et al., J. Biol. Chem. 271:10821–10826, 1996).
Systematic biochemical and genetic analyses of various mutants has allowed for the dissection of multiple functions of yeast Rad3. For example, mutation of yeast Rad3 at lysine-48 to arginine abolishes its ATPase and helicase activity but has no effect on the ability of the protein to bind ATP (Sung, P. et al., EMBO J. 7:3363–3369, 1988.) Mutations in RAD3 have also resulted in mitotic hyper-recombination without affecting the UV-sensitivity. These rem-1 and rem-2 mutations (for recombination/mutation) have been mapped to codons 237 and 661 (Montelone, B. et al., Genet. 119:289–301, 1988; Song, J M et al., J. Bacteriol. 172:6620–6630, 1990; Montelone B A and Malone R E, Yeast 10:13–27, 1994). Another RAD3 mutant (Gly-595 Arg) shows elevated levels of recombination between sequences shorter than 300 bp (Bailis, A et al., Mol. Cell. Biol. 15:3998–4008,1995). Finally, the rad3-1 allele has recently been shown to increase the efficiency of mismatch repair (Yang, Y. et al., Genet. 144:459–466, 1996).
Homologues of the S. cerevisiae RAD3 gene have been cloned from Schizosaccharomyces pombe, human, hamster, fish, and mouse (Reynolds P R et al., Nucleic Acid Res. 20:2327–2334,1992; Murray J M et al., Nucleic Acids Res. 20:2673–2678, 1992; Sung P et al., Nature 365:852–855, 1993; Weber Calif. et al., Mutat. Res. 324:147–152, 1993; Kirchner J M et al., Genomics 23:592–599, 1994; Walter R B et al., Genomics 10: 1083–1086, 1991; de Boer J et al., Cancer Res. 58:89–94, 1998). Recently, a Drosophila melanogaster sequence (Accession Number AF132140) and an Arabidopsis thaliana sequence (Accession Number AC005278) both showing similarity to RAD3 have been deposited in Genbank. The present invention describes a full-length cDNA sequence which encodes the maize orthologue of RAD3.
The modulation of Rad3 will provide for many advantages. One advantage involves the regulation of DNA repair and recombination. Enhancing DNA repair and DNA recombination will increase the efficiency with which heterologous nucleic acids are incorporated into the genomes of a target plant cell. Control of these processes has important implications in the creation of novel recombinantly engineered crops such as maize or soybean.
Another advantage to the modulation of Rad3 involves cell viability. RAD3 mutants have been found to be lethal in haploid cells (Naumovski, L., and E. C. Friedberg, Proc. Natl. Acad. Sci. 80:4818–4821, 1983). Thus, by reducing Rad3 levels in anther cells, development may cease, which may lead to a male sterile phenotype. Alternatively, if Rad3 expression in cell culture is modulated by the use of an inducible promoter, cell growth may be induced, thereby improving transformation. The present invention provides for these and other advantages.